Glutathione (GSH) and its associated enzymes comprise a major antioxidant system in brain. Neurodegenerative disease; including Parkinson's, ALS, Alzheimer's and Friedreich's ataxia involve oxidative damage and mitochondrial deficits. It is therefore, essential to develop an understanding of the roles served by GSH during mitochondrial impairment and oxidative insult. One little studied role for GSH is in protein-glutathione-mixed disulfide (PrSSG) formation, but has become of increasing interest as a mechanism for modulation of damage during oxidative stress. Previous work by us showed that during mitochondrial inhibition or oxidative stress, GSH is incorporated into protein to form PrSSG, a reversible reaction catalyzed by glutaredoxin (Grx). Importantly, we identified in brain mitochondria, functional Grx, an enzyme that could play a role in mitochondrial function or dysfunction during oxidative stress. A cytosolic form of Grx has been found in neurons. The overall objective of the current project is to expand our knowledge of the role and mechanism of protein thiolation and dethiolation during acute and chronic oxidative stress. The hypothesis to be examined is that during acute oxidative stress, PrSSG formation serves a reversible and protective function. In contrast, chronic long-term exposure to oxidative stress can result in impaired dethiolation that can contribute to neuronal damage. Aim 1 will investigate cellular Grx and PrSSG formation during acute and chronic mitochondrial inhibition to determine if dethiolation is altered and if so, the underlying mechanism, and if enhancing Grx activity modulates neuronal viability during mitochondria inhibition. GSH and PrSSG have been linked to the ubiquitin proteasomal pathway. This aim will also examine the effects of graded GSH depletion on this pathway under basal or oxidatively challenged conditions. Proteomics will be used to identify the major cellular proteins glutathionylated during oxidative stress Aim 2 will investigate mitochondrial Grx 8 PrSSG formation during mitochondrial impairment. We will expand on previous work during the past funding period to further examine the functional consequences of PrSSG formation in mitochondria and to determine in vivo the consequences of mitochondrial impairmenton mitochondrial Grx activity and PrSSG formation. Proteomics will be used to identify mitochondrial proteins that are glutathionylated during oxidative stress. Little is know regarding Grx in brain. Aim 3 will characterize cytosolic Smitochondrial Grx and PrSSG levels with respect to age, gender and region and will determine i there are differences in Grx activity between synaptic and nonsynaptic mitochondria. Grx has not been identified in astrocytes. This aim will also determine if astrocytes contain Grx or are capable of carrying out thiolation and dethiolation o proteins during oxidative stress. Overall, the project will generate novel information about a little studied role o GSH and its importance during mitochondrial impairment or oxidative stress. The studies will have relevance to neurodegenerative conditions or pathophysiological situations in which metabolism is compromised.